According to the development of recombinant DNA technology, medically and industrially useful proteins, which could have been obtained at only small amounts in natural conditions, could be produced at large amounts in bacteria, yeast, mold, animal, plant, and insect cells. However, in mass-producing target proteins in host cells, the cells recognize the target proteins as stress and thus produce molecular chaperones for their protection. The molecular chaperones are mostly heat shock proteins (HSPs) and present at least one in all cells.
In the case of E. coli, the HSPs include SecB, DnaK, DnaJ, GrpE, GroEL, GroES, IbpA, IbpB and the like, and play important roles in DNA duplication, RNA synthesis, protein synthesis, folding and recycling, and cell growth and division, etc. (LaRossa and Van Dyk, Mol. Microbiol., 5, 529-34, 1991). The most typical HSPs in the cytoplasm of E. coli are Dnak-DnaJ-GrpE and GroEL-GroES. DnaK is bound to the hydrophobic fragment of a denatured protein and hydrolyzed by DnaJ, and then, DnaK is recycled by GrpE while restoring the denatured protein to a native protein. This folding of the denatured protein is accelerated by GroEL-GroES.
Many studies have been conducted in attempts to improve the production of target proteins using HSPs. Studies that had been conducted until now can be broadly divided into a method of producing target proteins in soluble form and a method of producing target proteins in insoluble inclusion body form. The former method is characterized in that HSPs are simultaneously expressed to improve the production and activity of target proteins. Goloubinoff et al. have reported that the production of active foreign ribulose bisphosphate carboxylases (ribulose) was improved by GroEL and GroES (Goloubinoff et al., Nature, 337, 44-7, 1989). Furthermore, Georgiou and Valax have reported the use of GroEL and GroES, or DnaK and DnaJ, in the production of β-galactosidase (Georgiou and Valax, Curr. Opin. Biotechnol., 7, 190-7, 1996). In addition, Murby et al. have reported that the DnaK protein essential for the expression and secretion of cell's own proteins or target proteins was coexpressed with alkaline phosphatase or fusion proteins (Murby et al., Biotechnol., Appl. Biochem., 14, 336-46, 1991).
However, this system has the following several problems in the production of target proteins: (1) since HSPs proteins are simultaneously expressed in excessive amounts, the ratio of HSPs proteins in total protein is increased to 30-50%, thereby relatively reducing a capability for cells to maximally synthesize target proteins, (2) since two expression vectors are used, the stability of plasmids is reduced, (3) since the exact folding of target proteins is achieved by the cooperative action of many different molecular chaperones, this folding cannot be achieved with one or two kinds of chaperones (Langer et al., Nature, 356, 683-9, 1992). Recently, in an attempt to solve some of such problems, there has been developed a method where strains having a mutant in HSPs themselves are used. Baneyx et al. have reported that the production of target proteins was increased 2-4 fold by the use of a DnaK mutant (U.S. Pat. No. 5,552,301).
Meanwhile, the latter method is characterized in that the production of target proteins is enhanced by the use of strains deficient in a regulator rpoH for HSPs. The rpoH gene (sigma 32, “δ32”) mutant stains reduces the synthesis of HSPs involved in the folding and degradation of a denatured protein, and thus, are suitable for use in the production of proteins liable to be degreed by protease, or in producing target proteins in insoluble inclusion body form. Easton et al. have reported that bIGF2 (bovine insulin-like growth factor 2) as an inclusion body in the cytoplasm was produced at 20-25% by the use of the rpoH strains Easton et al., Gene, 101, 291-5, 1991). Moreover, Obukowicz et al. have reported that the production of target proteins was enhanced by using a new rpoH mutant strain (Obukowicz et al., Appl. Environ. Microbiol., 58, 1511-23, 1992). In addition, Goldberg et al. have reported that the degradation of target proteins was reduced by using a rpoH and lon mutant strain, thereby enhancing the production of target proteins (U.S. Pat. No. 4,758,512). However, this system has a fatal effect on cell growth in producing target proteins.
Up until now there has been no use of small heat shock proteins (sHSPs) in the production of target proteins. sHSPs are HSPs with a small molecular weight of 12-42 kDa, and are induced by heat, or stress such as the overproduction of target proteins, and also protect the denaturation of target proteins. sHSPs are present in all organisms ranging from eukaryotes to prokaryotes, and sHSPs, which have been found until now, include human sHSPs (HSP27, α- and β-crystallin), murine HSP25, Pisum sativum (pea) HSP18.1, Saccharomyces cerevisiae HSP26, Bradirhizobium japonicum sHSPs (HSPH, HSPB, HSPC, HSPF), Metlaznocoecus jannaschii HSP16.5, Synechococcus vulcanus HSP16, and Mycobacterium tuberculosi HSP16.3 (Studer and Narberhaus, J. Biol. Chem., 275, 37212-8, 2000).
Particularly, it was reported that ibpA and/or ibpB genes as inclusion body-associated proteins belonging to sHSPs binds to target proteins in producing target proteins in recombinant E. coli (Allen et al., J. Bacteriol., 174, 6938-47, 1992). Thus, it is obvious that the ibpA and/or ibpB genes belonging to sHSPs protect denatured proteins by stress in organisms, particularly bacteria.
A method of secreting target proteins into E. coli periplasm or culture medium has the following several advantages: (1) since periplasm or medium contains proteins at significantly lower amounts than cytoplasm, target proteins can be isolated and purified at high purity (Nossal et al., J. Biol. Chem., 241, 3055-62, 1966), (2) since target proteins secreted into periplasm or medium are isolated from cytoplasm where most of proteases exist the degradation of target proteins caused by cytoplasmic proteases can be prevented in advance, thereby increasing the yield of target proteins (Meerman and Georgiou, Ann. NZ Acad. Sci., 721, 292-302, 1994), (3) since periplasm is a more oxidized environment than cytoplasm, disulfide binding is more easily made and thus the correct folding of produced proteins is achieved, thereby remarkably reducing the formation of an inclusion body (Hockney, TIBTECH; 12, 456-63, 1994). However, cases can also occur where no secretion of target proteins is made or the secreted proteins are inactive.
Meanwhile, it can be advantageous that any proteins are produced in insoluble inclusion body form. By this time, many studies on the production of an inclusion body were conducted, but the exact mechanism of inclusion body formation is not yet established and any general relation is not yet found (Makrides, S C, Microbiol. Rev., 60, 512-38, 1996). The formation of the inclusion body varies depending on a host-vector system, and the characteristics, and culturing and expression conditions of proteins, and thus, can be found only by a test in the desired system.
Therefore, the present inventors have conducted extensive studies in an attempt to develop a strain system of increasing the production of target proteins using bacteria, and consequently, found that the use of bacteria from which ibpA and ibpB genes coding for inclusion body-associated proteins of E. coli were deleted provides an increase in secretory production and activity of target proteins, and the use of bacteria where the ibpA and/or ibpB genes coding for inclusion body-associated proteins of E. coli were amplified provides an increase in production of target proteins in the cytoplasm and also allows for the production of target proteins in inclusion body form. On the basis of these points, the present invention was perfected.